Underlying Role of Hydrophobic Environments in Tuning Metal Elements for Efficient Enzyme Catalysis.

The catalytic functions of metalloenzymes are often strongly correlated with metal elements in the active sites. However, dioxygen-activating nonheme quercetin dioxygenases (QueD) are found with various first-row transition-metal ions when metal swapping inactivates their innate catalytic activity. To unveil the molecular basis of this seemingly promiscuous yet metal-specific enzyme, we transformed manganese-dependent QueD into a nickel-dependent enzyme by sequence- and structure-based directed evolution. Although the net effect of acquired mutations was primarily to rearrange hydrophobic residues in the active site pocket, biochemical, kinetic, X-ray crystallographic, spectroscopic, and computational studies suggest that these modifications in the secondary coordination spheres can adjust the electronic structure of the enzyme-substrate complex to counteract the effects induced by the metal substitution. These results explicitly demonstrate that such noncovalent interactions encrypt metal specificity in a finely modulated manner, revealing the underestimated chemical power of the hydrophobic sequence network in enzyme catalysis.

Discovery and Characterization of Polymyxin-Resistance Genes pmrE and pmrF from Sediment and Seawater Microbiome.

Polymyxins are the last-line antibiotics used to treat Gram-negative pathogens. Thus, the discovery and biochemical characterization of the resistance genes against polymyxins are urgently needed for diagnosis, treatment, and novel antibiotic design. Herein, we report novel polymyxin-resistance genes identified from sediment and seawater microbiome. Despite their low sequence identity against the known pmrE and pmrF, they show in vitro activities in UDP-glucose oxidation and l-Ara4N transfer to undecaprenyl phosphate, respectively, which occur as the part of lipid A modification that leads to polymyxin resistance. The expression of pmrE and pmrF also showed substantially high MICs in the presence of vanadate ions, indicating that they constitute polymyxin resistomes. IMPORTANCE Polymyxins are one of the last-resort antibiotics. Polymyxin resistance is a severe threat to combat multidrug-resistant pathogens. Thus, up-to-date identification and understanding of the related genes are crucial. Herein, we performed structure-guided sequence and activity analysis of five putative polymyxin-resistant metagenomes. Despite relatively low sequence identity to the previously reported polymyxin-resistance genes, at least four out of five discovered genes show reactivity essential for lipid A modification and polymyxin resistance, constituting antibiotic resistomes.

Emergence of metal selectivity and promiscuity in metalloenzymes.

Metal coordination with proteinaceous ligands has greatly expanded the chemical toolbox of proteins and their biological roles. The structure and function of natural metalloproteins have been determined according to the physicochemical properties of metal ions bound to the active sites. Concurrently, amino acid sequences are optimized for metal coordination geometry and/or dedicated action of metal ions in proteinaceous environments. In some occasions, however, natural enzymes exhibit promiscuous reactivity with more than one designated metal ion, under in vitro and/or in vivo conditions. In this review, we discuss selected examples of metalloenzymes that bind various first-row, mid- to late-transition metal ions for their native catalytic activities. From these examples, we suggest that environmental, inorganic, and biochemical factors, such as bioavailability, native organism, cellular compartment, reaction mechanism, binding affinity, protein sequence, and structure, might be responsible for determining metal selectivity or promiscuity. The current work proposes how natural metalloproteins might have emerged and adapted for specific metal incorporation under the given circumstances and may provide insights into the design and engineering of de novo metalloproteins.

Integrative metagenomic and biochemical studies on rifamycin ADP-ribosyltransferases discovered in the sediment microbiome.

Antibiotic resistance is a serious and growing threat to human health. The environmental microbiome is a rich reservoir of resistomes, offering opportunities to discover new antibiotic resistance genes. Here we demonstrate an integrative approach of utilizing gene sequence and protein structural information to characterize unidentified genes that are responsible for the resistance to the action of rifamycin antibiotic rifampin, a first-line antimicrobial agent to treat tuberculosis. Biochemical characterization of four environmental metagenomic proteins indicates that they are adenosine diphosphate (ADP)-ribosyltransferases and effective in the development of resistance to FDA-approved rifamycins. Our analysis suggests that even a single residue with low sequence conservation plays an important role in regulating the degrees of antibiotic resistance. In addition to advancing our understanding of antibiotic resistomes, this work demonstrates the importance of an integrative approach to discover new metagenomic genes and decipher their biochemical functions.